Stay on target

Most cases of its use so far are fairly experimental; scientists can’t always predict precisely how DNA will be modified.

Maybe they’re just not looking hard enough.

Researchers at the Francis Crick Institute in London discovered a set of simple rules that determine the precision of CRISPR technology.

“Until now, editing genes with CRISPR has involved a lot of guesswork, frustration, and trial and error,” Crick group leader Paola Scaffidi said in a statement.

The team analyzed effects of CRISPR genome editing at 1,491 target sites across 450 genes in human cells, only to find the outcomes can be predicted based on certain guidelines.

(via Francis Crick Institute)

These so-called “rules,” according to Crick, depend on the fourth letter from the end of the guide RNA (gRNA), adjacent to the cutting site.

Synthetic molecules made up of around 20 genetic letters (A, T, C, G), gRNAs direct the Cas9 enzyme along the genome until it finds the region of interest, as explained by Crick.

Once it identifies a match, it sticks like Velcro, and the molecular scissors cut through the DNA—broken three letters from the end of the target sequence. Bits of genetic code are then inserted or deleted, seemingly haphazardly, when the cell attempts to repair the break.

If, according to the study, that all-important fourth letter is an A or T, there will be a very precise genetic insertion; a C leads to relatively precise deletion; and a G causes “many imprecise deletions.”

The moral of the complicated story: Avoid sites containing a G to make genome editing much more predictable.

“We were amazed to discover that the rules that determine the outcome of CRISPR human genome editing are so simple,” according to Anob Chakrabarti, Wellcome Trust clinical PhD fellow in the Crick’s Bioinformatics and Computational Biology lab and co-first author of the study.

“By bearing these rules in mind when designing our guide RNAs, we can maximize the chances of getting the desired outcome of a specific gene edit—which is particularly important in a clinical context,” he said.

How “open” or “closed” the target DNA is also affects the outcome. In that adding compounds that force DNA to open (therefore allowing Cas9 to scan the genome) prompted more efficient editing

This technique also appears to work regardless of the tissue of origin.

“The effects of CRISPR were thought to be unpredictable and seemingly random, but by analyzing hundreds of edits we were shocked to find that there are actually simple, predictable patterns behind it all,” according to Scaffidi.

“This will fundamentally change the way we use CRISPR,” she continued, “allowing us to study gene function with greater precision and significantly accelerating our science.”